NBC1在大鼠生后胰腺发育中的表达

目的:研究碳酸氢钠协同转运栽体(NBCl)在大鼠生后胰腺发育过程中的表达变化及细胞定位.方法:采用RT-PCR和Westem blot分别检测了NBC1核酸和蛋白在新生(PO)、P7、P14、P21和成年时期胰腺的表达情况,用Double fluorescence immunohistochemistry 分析了NBC1在P7、P14和成年时期腺泡和β细胞的定位表达.结果:在大鼠胰腺生后发育过程中,NBC1核酸、蛋白在P14时特异高表达,而在P7和成年最低;在腺泡基底侧膜和β细胞膜有阳性信号,且在成年胰腺中β细胞膜阳性信号较腺泡基底侧膜强.结论:NBC1在生后发育重塑旺盛期特异高表达,而在凋亡旺盛期和成年期表达最低.与腺泡细胞相比在成年期NBC1更集中于β细胞.提示NBC1在胰腺生后发育过程中不仅与胰岛结构重塑而且与胰腺功能发挥相关.     

大鼠胰腺发育不同阶段基因表达分析

目的:探讨大鼠胰腺发育不同阶段基因表达规律.方法:运用高通量基因芯片技术检测大鼠胰腺从胚胎12.5天(E12.5)到成年不同时期基因表达情况.结果:基因芯片所涵盖的基因59%在E12.5有表达,65%在15.5有表达,63%在E18.8有表达,53%在新生胰腺中有表达,38%在成年胰腺中有表达.E18.5相对于E15.5表达上调的基因中代谢相关的酶类有121条,占上调基因的18.2%,E18.5相对于E15.5表达下调的基因主要是一些转录因子和骨架蛋白.结论:在大鼠胰腺的发育过程中,早中期以细胞分化为主,而后期则是以细胞功能成熟为主.     

大鼠不同发育阶段胰腺Wnt5a的表达

目的:探讨大鼠胰腺不同发育阶段与胰岛形成和功能完善相关基因的表达趋势.方法:分离孕12.5d(E12.5)、E15.5d(E15.5)、18.5d(E18.5),新生(P0),生后14天(P14)、21d(P21)及成年(AP)大鼠胰腺组织,采用高密度寡核苷酸芯片对E12.5、E15.5、E18.5、P0和AP胰腺进行基因转录水平分析,并用RT-PCR验证wnt5a在不同发育时期的表达.结果:与胰岛功能相关基因多从胚胎18.5d开始出现明显高表达;与细胞迁移、聚集黏附相关基因多在胚胎18.5d高表达;WNT信号通路相关基因在胚胎15.5d表达量最高,wnt5a在胚胎15.5d表达量达到高峰.结论:wnt5a可能与胰岛形成、功能完善及出生后的胰腺重塑有关.     

大鼠胰腺发育中原癌基因c-met的表达及蛋白定位

目的:研究原癌基因c-met在大鼠胰腺发育不同阶段的表达及定位.方法:采用RT-PCR技术检测c-met基因在大鼠胰腺不同发育时期:孕15.5天(E15.5)和孕18.5天、新生、生后14天(P14)、P21及成年胰腺的表达.并用免疫组化技术对该基因编码的蛋白-肝细胞生长因子受体c-MET蛋白在胰腺发育不同阶段的定位进行分析.结果:c-met基因在E15.5、E18.5较成年特异性高表达.免疫组化结果显示该基因编码的蛋白c-MET在新生后的胰腺大量定位与胰岛细胞.结论:提示c-met可能在胰腺发育过程中起到调控作用,参与胰腺发育中新生后胰岛结构重塑过程.     

大鼠胰腺胚胎发育功能相关基因表达谱的分析

目的:研究大鼠胰腺胚胎发育不同阶段的基因表达谱,对比其功能相关基因随大鼠胰腺发育的变化.方法:采用显微分离及提取技术获得胚胎发育不同阶段胰腺组织并提取RNA,采用高密度寡核普酸芯片(Affemetrix芯片)对胚胎发育至第12.5天、15.5天、18.5天胚胎胰腺及成年胰腺进行基因转录水平分析,用生物信息学方法分析具体基因的表达情况.结果:胰腺的生物学功能尤其beta细胞功能相关基因insulin RNA,amylopsin RNA,GLUT-2 RNA等在胚胎15.5及18.5天显著高表达.结论:E15.5到E18.5直至出生是胰腺功能完善和成熟的阶段,这个时期以细胞功能成熟为主.     

大鼠不同发育时期胰腺相关蛋白的差异表达

探讨大鼠胰腺不同发育时期相关蛋白的差异表达,应用显微技术分离了大鼠孕15.5天,孕18.5天胚胎胰腺和新生鼠及成年鼠的胰腺,提取其蛋白质后,用固相pH梯度双向聚丙烯酰胺凝胶电泳和质谱分析等蛋白质组学方法,得到了4个不同发育时期的蛋白质表达谱.对其中的6个在孕18.5天胚胎胰腺中有高丰度表达,而在成年鼠胰腺中缺失的蛋白质点,4个在成年胰腺中特异表达的蛋白质点, 8个在成年胰腺中表达明显下调的蛋白质点和1个在成年中表达上调的点,进行了肽质量指纹分析和蛋白质鉴定,共获得18个点的肽质量指纹图.经BIOWORK等软件搜索大鼠非冗余蛋白质数据库来鉴定其身份,发现其中7个点为大鼠甲胎蛋白（AFP）、5个点为胰脂酶相关蛋白1前体、1个点为微管蛋白β、2个点为蛋白二硫异构酶、1个为FLN29基因产物的类似物、1个为胰蛋白酶V-A前体、1个为过氧化物氧化还原酶4.其中AFP为特异表达于大鼠胚胎期及新生期胰腺的蛋白质,在孕18.5天的胰腺中表达量最高,在成年胰腺中极低表达.对它们的功能和与胚胎胰腺代谢调节功能完善过程的可能关系进行了初步探讨.     

大鼠胰腺发育过程中AFP动态表达的意义

目的:探索大鼠胰腺发育过程中AFP动态表达的意义.方法:采用高密度寡核苷酸芯片对胚胎12.5天(E12.5)、E15.5、E18.5、初生和成年大鼠胰腺进行基因转录水平分析.结果:AFP显著高表达于E18.5大鼠胰腺,AFP在胚胎期的动态表达趋势与胰腺功能细胞标志物以及可能介导AFP促细胞增殖效应的有关信号系统成员的表达趋势一致.结论:AFP在大鼠胰腺发育过程中动态表达的生物学意义可能在于参与胰腺发育调控.     

Gm2052基因在小鼠胚胎发育中表达的初步研究

目的:研究预测的编码蛋白基因Gm2052在小鼠胚胎发育阶段的表达模式,为进一步了解该基因的功能奠定基础。方法:通过全胚胎原位杂交技术、组织切片原位杂交技术及半定量RT-PCR方法,对预测的Gm2052基因在小鼠胚胎发育中后期及在新生小鼠中的表达情况进行初步分析。结果:全胚胎原位杂交显示,在E10.5小鼠胚胎中,Gm2052仅在脑中表达;当小鼠胚胎发育至E13.5时,Gm2052在脑、舌、肺、肝脏、胰腺等组织中均有表达。半定量RT-PCR结果显示,在小鼠胚胎中后期(E15.5和E18.5)及新生小鼠(出生后第9 d)中,Gm2052呈动态表达模式。结论:预测基因Gm2052与小鼠脑的发育密切相关,并可能参与小鼠肺、肝脏及胰腺等主要脏器胚期的发育。     

AI429618基因在小鼠胚期表达的研究

目的:通过对与小鼠胚胎发育相关的新基因AI429618表达模式的初步分析为揭示小鼠胚胎发育机理提供研究基础.方法:利用Northern-blot和原位杂交方法对该基因进行表达谱分析.结果:Northern结果表明该基因在E12.5,E.15.5,E18.5三个时期都有所表达,并且在E12.5的小鼠胚胎中处于一个相对较高的转录水平,E15.5表达骤降并且基本上与E18.5(略高)持平;原位杂交结果显示E9.5,E10.5的小鼠胚胎中这一基因的表达集中在端脑、中脑、后脑、腮弓、前肢芽以及尾芽,E15.5的切片原位杂交中这一基因的表达信号在胸腺,肺,肝,肾,小肠中极为显著.结论:AI429618基因在小鼠胚胎发育期有着持续广泛的表达,可能对胚胎的正常发育起着重要的调控作用.     

大鼠胰腺发育阶段Mesothelin的表达

目的:研究Mesothenlin在大鼠胰腺发育阶段的表达和细胞定位。方法:运用RT-PCR和Western Blot技术分别检测Mesothenlin在大鼠胰腺发育阶段的mRNA和蛋白表达水平;运用免疫荧光检测不同时期Mesothenlin在胰腺的组织细胞学定位。结果:RT-PCR结果显示E18.5 Mesothelin mRNA的表达水平显著增高,至P14达到高峰,成年较低。Western Blot结果显示其蛋白表达趋势与mRNA完全相同。免疫荧光结果显示在不同发育时期Mesothenlin与胰岛β细胞和间充质细胞共表达。结论:Mesothenlin在大鼠胚胎胰岛形成及生后结构重塑中出现显著性高表达,并表达于胰岛β细胞和间充质细胞。     

C-met及相关基因在m大鼠胰腺发育功能完善中的表达

目的：研究原癌基因c—met及其相关基因在大鼠胰腺发育及细胞功能完善过程中的表达。方法：采用高密度寡核苷酸芯片（Affymetrix芯片）对孕12．5（E12．5）、E15．5和E18．5、新生、成年胰腺进行基因转录水平分析,并用RT—PCR验证基因在大鼠胰腺不同发育时期的表达。结果：c—met基因在E15．5、E18．5较成年特异高表达。芯片中c—met转录调控基因和信号传导通路相关基因的表达趋势与c—met高度相似。RT—PCR（所用引物设计区域与芯片相同）验证,c—met表达趋势与芯片结果相符;与芯片c—met探针所用引物不同RT—PCR,结果却在各发育阶段呈现与芯片不同的表达趋势。结论：提示c—met可能在胰腺发育细胞功能完善的关键阶段起调控作用,参与胚胎胰腺发育中晚期细胞功能完善的信号传导过程。并且c—met在胰腺发育中发挥作用有可能存在不同转录本。     

Alpha-fetoprotein is dynamically expressed in rat pancreas during development

To identify proteins involved in pancreatic development, we used a differential proteomics approach by comparing pancreatic extracts from four biologically significant stages of development: embryonic day (E) 15.5, E18.5, postnatal (P) days 0 and adult. By two-dimensional gel electrophoresis (2D-E) and MALDI-TOF MS (Matrix Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) following database searching and protein annotation, 15 proteins were identified as being differently expressed in the pancreas between the four phases. The expression pattern and the localization of alpha-fetoprotein (AFP), one of significant changed proteins observed, were further determined. Four isoforms of AFP (72 kDa, 60 kDa, 48 kDa and 37 kDa) were found by Western blotting in the pancreas tested, most of them showed a stronger signal in E18.5 followed by a steady decrease and only a 60-kDa isoform was detected in the adult pancreas. Immunolocalization for AFP revealed that a positive reactivity was detectable at E15.5 pancreas, became stronger in the cytoplasm of mesenchyme cells at E18.5, and declined after birth to a nearly undetectable level in adults. The dynamic expression of AFP in rat pancreas from different stages indicates that AFP might be involved in some aspects of pancreatic development.     

BTG2基因在大鼠胚胎胰腺不同发育阶段的表达

利用高密度寡核苷酸芯片技术对大鼠胚胎胰腺发育中晚期(E12．5,E18．5,E15．5)调节内外分泌部细胞发育分化及功能代谢的基因的表达趋势进行研究。并用RT-PCR进行验证。用获得的基因信息对:NCBI等公共数据库进行检索,结果发现对细胞的分化、增殖和凋亡起调节作用的BTG2基因在大鼠胚胎胰腺E12．5、E15．5、E18．5天及成年、新生大鼠胰腺中均有表达,且E18．5天的表达量高于其他时期5倍多。推测BTG2可能在大鼠胚胎胰腺内外分泌细胞分化发育的不同阶段起到了促进作用,并参与胚胎胰腺发育晚期的功能代谢完善过程。     

Ontogenetic expression of chromogranin A and its derived peptides, WE-14 and pancreastatin, in the rat neuroendocrine system

 The ontogenetic expression of chromogranin A (CgA) and its derived peptides, WE-14 and pancreastatin (PST), was studied in the rat neuroendocrine system employing immunohistochemical analysis of fetal and neonatal specimens from 12.5-day embryos (E12.5), to 42-day postnatal (P42) rats. CgA immunostaining was first detected in endocrine cells of the pancreas, stomach, intestine, adrenal gland and thyroid at E13.5, E14.5, E15.5, E15.5 and E18.5, respectively. PST-like immunoreactivity was detected in endocrine cells of the pancreas at E13.5, stomach, intestine at E15.5, adrenal gland at E17.5 and thyroid at E18.5. WE-14 immunoreactivity was first observed in the immature pancreas at E15.5, mucosal cells of the stomach at E15.5, scattered chromaffin cells in the immature adrenal gland and mucosal cells of the intestine at E17.5 and thyroid parafollicular cells at E18.5. These data confirm that the translation of the CgA gene is regulated differentially in various neuroendocrine tissues and, moreover, suggests that the posttranslational processing of the molecule is developmentally controlled. Accepted: 18 October 1996     

Thyroid hormones promote endocrine differentiation at expenses of exocrine tissue

Diabetes is caused by loss or dysfunction of pancreatic beta cells. Generation of beta cells in vitro is a promising strategy to develop a full-scale cell therapy against diabetes, and the development of methods without gene transfer may provide safer protocols for human therapy. Here we show that thyroid hormone receptors are expressed in embryonic murine pancreas. Addition of the thyroid hormone T3 in an ex vivo culture model of embryonic (E12.5) dorsal pancreas, mimicking embryonic pancreatic development, promoted an increase of ductal cell number at expenses of the acinar compartment. Double labeled cells expressing specific markers for ductal and acinar cells were observed, suggesting cell reprogramming. Increased mRNA levels of the pro-endocrine gene Ngn3 and an increased number of beta cells were detected in cultures treated previously with T3 suggesting that ductal cells promoted by T3 can subsequently differentiate into endocrine cells. So, indirectly, T3 induced endocrine differentiation. Moreover, T3 induced the expression of the pro-endocrine gene Ngn3 in the acinar 266-6 cell line. The pro-endocrine effect of T3 in the pancreatic explants and in the acinar cell line, was abrogated by the Akt inhibitor Ly294002 indicating the involvement of Akt signaling in this process. Altogether we show numerous evidences that define T3 as a promising candidate to generate endocrine cells from exocrine tissue, using ectopically gene expression free protocols, for cell therapy against diabetes.